title: Identification of amino acid residues of matrix metalloproteinase-7 essential for binding to cholesterol sulfate.
abstract: Matrix metalloproteinase-7 (MMP-7; matrilysin) induces homotypic adhesion of colon cancer cells by cleaving cell surface protein(s) and enhances their metastatic potential. Our previous study (Yamamoto, K., Higashi, S., Kioi, M., Tsunezumi, J., Honke, K., and Miyazaki, K. (2006) J. Biol. Chem. 281, 9170-9180) demonstrated that binding of MMP-7 to cell surface cholesterol sulfate (CS) is essential for the cell membrane-associated proteolytic action of the protease. To determine the region of MMP-7 essential for binding to CS, we constructed chimeric proteases consisting of various parts of MMP-7 and those of the catalytic domain of MMP-2; the latter protease does not have an affinity for CS. Studies of these chimeric proteases and other mutants of MMP-7 revealed that Ile29, Arg33, Arg51, and Trp55, in the internal sequence, and the C-terminal three residues corresponding to residues 171-173 of MMP-7 are essential for binding to CS. An MMP-7 mutant, which had the internal 4 residues at positions 29, 33, 51, and 55 of MMP-7 replaced with the corresponding residues of MMP-2 and the C-terminal 3 residues deleted, had essentially no affinity for CS. This mutant and wild-type MMP-7 showed similar proteolytic activity toward fibronectin, whereas the mutant lacked the ability to induce the colon cancer cell aggregation. In the three-dimensional structure of MMP-7, the residues essential for binding to CS are located on the molecular surface in the opposite side of the catalytic cleft of the protease. Therefore, it is assumed that the active site of MMP-7 bound to cell surface is directed outside. We speculate that the direction of the cell-bound MMP-7 makes it feasible for the protease to cleave its substrates on cell surface.
authors.affiliation: International Graduate School of Arts and Sciences, Yokohama City University, Yokohama 244-0813, Japan. shigashi@yokohama-cu.ac.jp
authors.forename: Shouichi
authors.initials: S
authors.lastname: Higashi
authors.forename: Miwa
authors.initials: M
authors.lastname: Oeda
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Kaoru
authors.initials: K
authors.lastname: Miyazaki
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D002788:Cholesterol Esters / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D020783:Matrix Metalloproteinase 7 / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010449:Peptide Mapping / Q000379:methods; D011485:Protein Binding / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D002788:Cholesterol Esters; D011993:Recombinant Fusion Proteins; C060932:MMP7 protein, human; D020783:Matrix Metalloproteinase 7; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; C007045:cholesteryl sulfate